Interference microscopes are known from practical use. EP 0 491 289 A1, for example, discloses a double-confocal scanning microscope or 4π microscope in which a specimen is illuminated in point-like fashion by two microscope objectives arranged opposite one another. As a result of this double-sided illumination of the specimen and/or the double-sided detection of the light coming from the specimen, an interference pattern is created with which an increase in axial resolution can be achieved.
U.S. Pat. No. 4,621,911 discloses a standing wave field microscope in which a standing wave field or interference pattern serving to illuminate a specimen is formed by the superposition of two light beams proceeding in collimated fashion. This standing wave field has planes of equal illumination intensity oriented parallel to the focal plane of the microscope objectives, the illumination intensity varying from a maximum illumination intensity value to a minimum illumination intensity value, and the alternating illumination variation being continued periodically along the optical axis of the microscope objectives. With this interferometric illumination method, fluorescent specimens can be excited to fluoresce in accordance with the illumination pattern, thereby also allowing an axial resolution improvement to be achieved.
U.S. Pat. No. 5,671,085 discloses an I2M, I3M, or I5M microscope in which a specimen is also excited to fluoresce with a bright-field incident illumination through two microscope objectives arranged opposite one another. Here as well, the illuminating light and/or detected light can be caused to interfere, thereby again making it possible to achieve axial resolution improvements.
Very generally, interference microscopes comprise an illuminating beam path of at least one light source, as well as a detected beam path of at least one detector. In the aforementioned interference microscopes, two objectives are arranged on either side of the specimen plane, the objectives being directed toward one another. At least one beam splitter for distributing the illuminating light to the objectives, and a beam combiner for combining the detecting light coming from the objectives, is provided in the illuminating/detected beam paths. The beam splitter and the beam combiner can be configured as one and the same component. Specimens specifically stained with fluorescent markers, in particular biological specimens, are usually examined with the aforementioned interference microscopes. In this context the light of the light source is used to excite the fluorescent markers, and only that fluorescent light is detected by the detector.
Because of their interferometric construction and the small dimensions of the objective focus, interference microscopes of the species are highly susceptible to shock, vibration, and thermal expansion. The equalization of optical path length differences between the interferometer beam path segments is, in particular, a critical influencing variable for successful operation of an interference microscope. The optical path length differences must be so small that, on the one hand, the illuminating light passing through the two interferometer beam path segments can interfere; i.e. the optical path length difference between the two interferometer beam path segments must be smaller than the coherence length of the illuminating light. On the other hand, the two interferometer beam path segments must be equalized with one another in terms of optical path length difference in such a way that constructive interference is present in the specimen region of the interference microscope.
In interference microscopes hitherto implemented, alignment of the interference beam path segments is performed, in practice, on the basis of detections of individual specimen regions. For example, an axial optical section through a point-like or linear specimen is acquired, and alignment of the interference microscope is performed on the basis of its intensity signal profile. From the axial intensity signal profile, conclusions can be drawn regarding the illumination conditions actually present in the specimen region, i.e. as to whether constructive or destructive interference is present. This alignment is complex, and must be performed individually by the operator of the interference microscope. In addition, a great deal of experience on the part of the operator is indispensable for successful alignment, so that ultimately, interference microscopes of the species can be used only by a small group of operators; this has heretofore impeded wide distribution of the interference microscopes discussed above.